The goal of this project is to identify the cis and trans-acting elements involved in the coordinate transcriptional regulation of a large number of unlinked genes encoding amino acid biosynthetic enzymes in the yeast Saccharomyces cerevisiae. The minimal cis-acting regulatory element at one such gene, HIS4, has been identified by the construction of HIS4-CYC1 promoter fusions that place the CYC1 gene under general amino acid control. A synthetic 14 base pair fragment from the HIS4 promoter, containing a single copy of a short nucleotide sequence found repeated upstream from HIS4 and other co-regulated genes, is sufficient to confer the general control regulatory response. Molecular analysis of the GCN4 gene, which encodes a trans-acting positive regulator of general control, has shown that GCN4 expression is itself regulated by amino acid starvation and that this regulation is mediated by other general control trans-acting factors. Moreover, GCN regulation occurs at the translational level and is exerted by sequences found in the 600 nucleotide 5' leader of its mRNA. The leader control region contains four small open-reading frames of 2-3 codons each. The repression exerted by these sequences appears to be mediated by the GCD1 product, a known repressor of amino acid biosynthetic enzymes. Repression is released during amino acid starvation by the GCN2 and GCN3 products, known activators of amino acid control and antagonists of GCD1. In vitro mutagenesis will now be used to identify more precisely the leader sequences controlling GCN4 translation, and in vivo mutant isolation will be conducted to identify trans-acting factors required for GCN4 translational repression. The GCN4 protein is the best candidate for the transcriptional activator that interacts with the repeated sites of positive control in the co-regulated structural genes. Isolation of the GCN4 protein will be undertaken to test this possibility in vitro.